The oligomeric state of the active Vps4 AAA ATPase.
The oligomeric state of the active Vps4 AAA ATPase.
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DOI：
10.1016/j.jmb.2013.09.043
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发表时间：
2014-02-06
影响因子：
5.6
通讯作者：
Hill CP
中科院分区：
文献类型：
--
作者：
Monroe N;Han H;Gonciarz MD;Eckert DM;Karren MA;Whitby FG;Sundquist WI;Hill CP
细胞ESCRT途径驱动膜收缩朝向胞质溶胶，并在胞质分裂、内体分选和许多包膜病毒（包括HIV）的释放期间影响膜分裂。该途径的一个组成部分，AAA ATP酶Vps4，为途径进展提供能量。虽然已经确定Vps4作为寡聚体发挥作用，但亚基化学计量和功能酶的其他基本特征尚不清楚。到目前为止，仅在ATP存在下表征了Vps4的步行者B突变体的高阶寡聚体。在这里，我们报告说，虽然一些突变Vps4蛋白形成十二聚体组装，活性野生型S。酿酒酵母和酿酒酵母。如通过尺寸排阻色谱法和平衡分析超离心法测定的，在ATP和ADP存在下，solfataricus Vps4酶可以形成六聚体。Vta1p激活剂结合六聚体酵母Vps4p而不改变Vps4p的寡聚体状态，这意味着活性Vta1p：Vps4p复合物也包含单个六聚体环。此外，我们报告了两种不同的古细菌Vps4同源物的晶体结构，其结构和晶格相互作用表明一种保守的寡聚化模式。通过诱变破坏所提出的六聚化界面，消除了古细菌Vps4蛋白的ATP酶活性，并阻断了S.啤酒。这些数据挑战了流行的模型，即活性Vps4是一个双环十二聚体，并认为，像其他I型AAA ATP酶，Vps4功能作为一个单环与六个亚基。
The cellular ESCRT pathway drives membrane constriction toward the cytosol and effects membrane fission during cytokinesis, endosomal sorting, and the release of many enveloped viruses, including HIV. A component of this pathway, the AAA ATPase Vps4, provides energy for pathway progression. Although it is established that Vps4 functions as an oligomer, subunit stoichiometry and other fundamental features of the functional enzyme are unclear. Higher-order oligomers have thus far only been characterized for a Walker B mutant of Vps4 in the presence of ATP. Here, we report that although some mutant Vps4 proteins form dodecameric assemblies, active wild-type S. cerevisiae and S. solfataricus Vps4 enzymes can form hexamers in the presence of ATP and ADP, as assayed by size exclusion chromatography and equilibrium analytical ultracentifugation. The Vta1p activator binds hexameric yeast Vps4p without changing the oligomeric state of Vps4p, implying that the active Vta1p:Vps4p complex also contains a single hexameric ring. Additionally, we report crystal structures of two different archaeal Vps4 homologs, whose structures and lattice interactions suggest a conserved mode of oligomerization. Disruption of the proposed hexamerization interface by mutagenesis abolished the ATPase activity of archaeal Vps4 proteins and blocked Vps4p function in S. cerevisiae. These data challenge the prevailing model that active Vps4 is a double ring dodecamer, and argue that, like other type I AAA ATPases, Vps4 functions as a single ring with six subunits.